pvuii hf  (New England Biolabs)


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    Structured Review

    New England Biolabs pvuii hf
    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using <t>HpaI</t> and <t>PvuII</t> ensured that any residual partial digest products
    Pvuii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 33 article reviews
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    Images

    1) Product Images from "Recombination regulator PRDM9 influences the instability of its own coding sequence in humans"

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1220813110

    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products
    Figure Legend Snippet: De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Techniques Used: Mutagenesis, Amplification

    2) Product Images from "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii"

    Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27004-1

    Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.
    Figure Legend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

    Techniques Used: Sequencing, Transfection

    3) Product Images from "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii"

    Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27004-1

    Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.
    Figure Legend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

    Techniques Used: Sequencing, Transfection

    4) Product Images from "A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy"

    Article Title: A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab770

    High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.
    Figure Legend Snippet: High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.

    Techniques Used: In Vivo

    5) Product Images from "Antimicrobial Resistance in Escherichia coli and Resistance Genes in Coliphages from a Small Animal Clinic and in a Patient Dog with Chronic Urinary Tract Infection"

    Article Title: Antimicrobial Resistance in Escherichia coli and Resistance Genes in Coliphages from a Small Animal Clinic and in a Patient Dog with Chronic Urinary Tract Infection

    Journal: Antibiotics

    doi: 10.3390/antibiotics9100652

    Restriction profile of phage DNA using PvuII . This gel documents 12 restricted phages in which three restriction profiles could be distinguished. Slot 1 and 14 molecular weight marker, slot 2, 3, 7, 8, 9 restriction profile 1 (RP1), slot 4, 5, 6, 10, 11, 13 restriction profile 2 (RP2), slot 12 restriction profile 3 (RP3).
    Figure Legend Snippet: Restriction profile of phage DNA using PvuII . This gel documents 12 restricted phages in which three restriction profiles could be distinguished. Slot 1 and 14 molecular weight marker, slot 2, 3, 7, 8, 9 restriction profile 1 (RP1), slot 4, 5, 6, 10, 11, 13 restriction profile 2 (RP2), slot 12 restriction profile 3 (RP3).

    Techniques Used: Molecular Weight, Marker

    6) Product Images from "A novel zinc-finger nuclease platform with a sequence-specific cleavage module"

    Article Title: A novel zinc-finger nuclease platform with a sequence-specific cleavage module

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1112

    Kinetic analysis of the ZF-PvuII fusion enzymes cleaving an addressed substrate [black triangle; 250 bp PCR product containing either the tripartite (Z12P12Z) or bipartite (P12Z) target sites] and an unaddressed substrate [open diamond; 450 bp PCR product containing a single PvuII site (P)] in competition in a near equimolar stoichiometry (20 nM addressed substrate/20 nM unaddressed substrate/18 nM enzyme). The gel electrophoretic analysis of samples withdrawn from the incubation mixture at defined time intervals is shown as an insert of the activity versus time profile. ( A ) Preferential DNA cleavage (addressed versus unaddressed) by ZF-PvuII G46 (Z12P12Z versus P). ( B ) Preferential cleavage by ZF-scPvuII G46 (P12Z versus P). ( C ) Preferential DNA cleavage by ZF-PvuII G46/F94 (Z12P12Z versus P). ( D ) Preferential DNA cleavage by ZF-PvuII G46/A83/F94 (Z12P12Z versus P).
    Figure Legend Snippet: Kinetic analysis of the ZF-PvuII fusion enzymes cleaving an addressed substrate [black triangle; 250 bp PCR product containing either the tripartite (Z12P12Z) or bipartite (P12Z) target sites] and an unaddressed substrate [open diamond; 450 bp PCR product containing a single PvuII site (P)] in competition in a near equimolar stoichiometry (20 nM addressed substrate/20 nM unaddressed substrate/18 nM enzyme). The gel electrophoretic analysis of samples withdrawn from the incubation mixture at defined time intervals is shown as an insert of the activity versus time profile. ( A ) Preferential DNA cleavage (addressed versus unaddressed) by ZF-PvuII G46 (Z12P12Z versus P). ( B ) Preferential cleavage by ZF-scPvuII G46 (P12Z versus P). ( C ) Preferential DNA cleavage by ZF-PvuII G46/F94 (Z12P12Z versus P). ( D ) Preferential DNA cleavage by ZF-PvuII G46/A83/F94 (Z12P12Z versus P).

    Techniques Used: Polymerase Chain Reaction, Incubation, Activity Assay

    ZF-PvuII G46/A83/F94 mediated DNA cleavage in mammalian cells. ( A ) Schematic of the ZF-PvuII G46/A83/F94 target sites. The addressed Z12P12Z target site harbors an inverted repeat of ZF-binding sites separated by 12-bp spacer sequences that flank a central PvuII site. The unaddressed target site 21P21 is structured identically but lacks the ZF-binding sites. ( B ) Expression levels of ZF-PvuII G46/A83/F94 and PvuII G46/A83/F94. Cell lysates of transfected HEK293T cells were probed with antibodies against HA-tag or EGFP. ( C and D ) Cleavage activity in cellula . Cleavage of target plasmids in transfected HEK293T cells was assessed by detecting nuclease-induced mutations due to imperfect repair of DNA DSBs by NHEJ. PCR fragments encompassing the target site were either subjected to digestion with the mismatch-sensitive T7 endonuclease 1 (C) or PvuII (D).
    Figure Legend Snippet: ZF-PvuII G46/A83/F94 mediated DNA cleavage in mammalian cells. ( A ) Schematic of the ZF-PvuII G46/A83/F94 target sites. The addressed Z12P12Z target site harbors an inverted repeat of ZF-binding sites separated by 12-bp spacer sequences that flank a central PvuII site. The unaddressed target site 21P21 is structured identically but lacks the ZF-binding sites. ( B ) Expression levels of ZF-PvuII G46/A83/F94 and PvuII G46/A83/F94. Cell lysates of transfected HEK293T cells were probed with antibodies against HA-tag or EGFP. ( C and D ) Cleavage activity in cellula . Cleavage of target plasmids in transfected HEK293T cells was assessed by detecting nuclease-induced mutations due to imperfect repair of DNA DSBs by NHEJ. PCR fragments encompassing the target site were either subjected to digestion with the mismatch-sensitive T7 endonuclease 1 (C) or PvuII (D).

    Techniques Used: Binding Assay, Expressing, Transfection, Activity Assay, Non-Homologous End Joining, Polymerase Chain Reaction

    7) Product Images from "TALE-PvuII Fusion Proteins - Novel Tools for Gene Targeting"

    Article Title: TALE-PvuII Fusion Proteins - Novel Tools for Gene Targeting

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082539

    Engineered highly specific endonucleases that can be used for gene targeting by introducing a double-strand break into a complex genome and thereby stimulating homologous recombination. With the exception of engineered homing endonucleases (“meganucleases”) in which the function of DNA binding and DNA cleavage is present in the same polypeptide chain [ 77 ], the other engineered nucleases consist of separate DNA-binding (green) and DNA-cleavage (blue) modules. Zinc finger nucleases and TALE nucleases usually have the non-specific cleavage domain of the restriction endonuclease FokI as DNA-cleavage module, but as shown recently and in the present paper the restriction endonuclease PvuII can also be used for this purpose [ 54 ]. PvuII has also been employed in TFO-linked nucleases [ 49 ] and in protein fusions (with catalytically inactive I-SceI) [ 53 ] as DNA-cleavage module. Zinc finger nucleases, TALE nucleases and TFO-linked nucleases are programmable, as are the RNA-mediated nucleases [ 36 ] [modified after [ 3 ]] .
    Figure Legend Snippet: Engineered highly specific endonucleases that can be used for gene targeting by introducing a double-strand break into a complex genome and thereby stimulating homologous recombination. With the exception of engineered homing endonucleases (“meganucleases”) in which the function of DNA binding and DNA cleavage is present in the same polypeptide chain [ 77 ], the other engineered nucleases consist of separate DNA-binding (green) and DNA-cleavage (blue) modules. Zinc finger nucleases and TALE nucleases usually have the non-specific cleavage domain of the restriction endonuclease FokI as DNA-cleavage module, but as shown recently and in the present paper the restriction endonuclease PvuII can also be used for this purpose [ 54 ]. PvuII has also been employed in TFO-linked nucleases [ 49 ] and in protein fusions (with catalytically inactive I-SceI) [ 53 ] as DNA-cleavage module. Zinc finger nucleases, TALE nucleases and TFO-linked nucleases are programmable, as are the RNA-mediated nucleases [ 36 ] [modified after [ 3 ]] .

    Techniques Used: Homologous Recombination, Binding Assay, Zinc-Fingers, Modification

    Analysis of competition cleavage experiments with AvrBs3-PvuII fusion proteins. ( A ) Competition cleavage experiments with AvrBs3-28-L-PvuII T46G under physiological ionic strength. Shown is the cleavage pattern with supercoiled plasmid DNA with an addressed site (8 nM) in competition with a PCR fragment (unP) with an unaddressed site (32 nM). The experiment was carried out with a variable excess of enzyme over plasmid substrate (0.25 to 40-fold). The enzyme shows complete cleavage of the addressed substrate but no cleavage of the unaddressed substrate, even in an overnight incubation with a 40-fold excess of enzyme over the addressed plasmid substrate (8 nM) and 10-fold excess over the unaddressed PCR substrate (32 nM). The brackets indicate the positions where one would expect the products of cleavage of the unaddressed PCR substrate. oc, open circle; lin, linearized; sc, supercoiled. ( B ) Quantitative determination of the preference of AvrBs3-28-L-PvuII T46G for an addressed (T3-6bp-P-6bp-T3) over an unaddressed site (-P-). The reactions were performed in triplicate under physiological conditions with 20 nM enzyme and 20 nM addressed substrate (squares) and unaddressed substrate (circles), both PCR fragments were radioactively labelled with [α 32 P]dATP. The insert shows the primary data: the electrophoretic analysis of the cleavage reaction products using an Instant Imager. From the fit, a cleavage preference of > 34,000-fold was determined.
    Figure Legend Snippet: Analysis of competition cleavage experiments with AvrBs3-PvuII fusion proteins. ( A ) Competition cleavage experiments with AvrBs3-28-L-PvuII T46G under physiological ionic strength. Shown is the cleavage pattern with supercoiled plasmid DNA with an addressed site (8 nM) in competition with a PCR fragment (unP) with an unaddressed site (32 nM). The experiment was carried out with a variable excess of enzyme over plasmid substrate (0.25 to 40-fold). The enzyme shows complete cleavage of the addressed substrate but no cleavage of the unaddressed substrate, even in an overnight incubation with a 40-fold excess of enzyme over the addressed plasmid substrate (8 nM) and 10-fold excess over the unaddressed PCR substrate (32 nM). The brackets indicate the positions where one would expect the products of cleavage of the unaddressed PCR substrate. oc, open circle; lin, linearized; sc, supercoiled. ( B ) Quantitative determination of the preference of AvrBs3-28-L-PvuII T46G for an addressed (T3-6bp-P-6bp-T3) over an unaddressed site (-P-). The reactions were performed in triplicate under physiological conditions with 20 nM enzyme and 20 nM addressed substrate (squares) and unaddressed substrate (circles), both PCR fragments were radioactively labelled with [α 32 P]dATP. The insert shows the primary data: the electrophoretic analysis of the cleavage reaction products using an Instant Imager. From the fit, a cleavage preference of > 34,000-fold was determined.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Incubation

    Activity and toxicity of TALE-PvuII fusion proteins in human cells. ( A ) PCR was performed with the plasmid from the HEK293 cells resulting in a DNA fragment of 517 bp. * indicates the cleavage site of PvuII. ( B ) Analysis of the PCR product (14.5 nM) after digestion with 20 U of PvuII for 1 h. A cleavage-resistant band indicates the loss of the PvuII site by NHEJ and confirms the activity of the TALE-PvuII fusion proteins. ( C ) Cell toxicity of the PvuII-based TALENs. After co-transfection of a mCherry expression plasmid, cell survival rate was calculated as the decrease in the number of mCherry-positive cells from day 2 to day 5 by flow cytometry, normalized to cells transfected with an I-SceI expression vector. * Statistically significant differences in toxicities between I-SceI and TALE-PvuII fusion proteins are indicated (P-values) .
    Figure Legend Snippet: Activity and toxicity of TALE-PvuII fusion proteins in human cells. ( A ) PCR was performed with the plasmid from the HEK293 cells resulting in a DNA fragment of 517 bp. * indicates the cleavage site of PvuII. ( B ) Analysis of the PCR product (14.5 nM) after digestion with 20 U of PvuII for 1 h. A cleavage-resistant band indicates the loss of the PvuII site by NHEJ and confirms the activity of the TALE-PvuII fusion proteins. ( C ) Cell toxicity of the PvuII-based TALENs. After co-transfection of a mCherry expression plasmid, cell survival rate was calculated as the decrease in the number of mCherry-positive cells from day 2 to day 5 by flow cytometry, normalized to cells transfected with an I-SceI expression vector. * Statistically significant differences in toxicities between I-SceI and TALE-PvuII fusion proteins are indicated (P-values) .

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Plasmid Preparation, Non-Homologous End Joining, TALENs, Cotransfection, Expressing, Flow Cytometry, Cytometry, Transfection

    Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins. ( A ) and ( B ) Comparison of the cleavage rates of selected AvrBs3-PvuII fusion proteins (as indicated) under low ionic strength: 76 mM (20 mM Tris-Ac, 50 mM K-Ac, 2 mM Mg-Ac, pH 7.5). In the top row the cleavage of the addressed substrate (T3-6bp-P-6bp-T3) is shown, in the bottom row that of the unaddressed substrate (-P-). All cleavage experiments were done with 8 nM DNA and 8 nM enzyme. ( C ) Comparison of the cleavage rates of an unaddressed substrate by selected AvrBs3-PvuII fusion proteins (as indicated) under physiological ionic strength: 143 mM (20 mM Tris-Ac, 120 mM K-Ac, 1 mM Mg-Ac, pH 7.5). The experiments were done with an excess of enzyme, the TALE-scPvuII fusion protein (top, 60 nM enzyme, 6 nM DNA) shows a higher cleavage activity with an unaddressed substrate (-P-) than the homodimeric TALE-PvuII T46G fusion protein (bottom, 80 nM enzyme, 8 nM DNA). See the appearance of nicked and linearized DNA with AvrBs3-28-L-scPvuII T46G . There is no nicking or cleavage detectable of the unaddressed substrate with AvrBs3-28-L-PvuII T46G . oc, open circle; lin, linearized; sc, supercoiled.
    Figure Legend Snippet: Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins. ( A ) and ( B ) Comparison of the cleavage rates of selected AvrBs3-PvuII fusion proteins (as indicated) under low ionic strength: 76 mM (20 mM Tris-Ac, 50 mM K-Ac, 2 mM Mg-Ac, pH 7.5). In the top row the cleavage of the addressed substrate (T3-6bp-P-6bp-T3) is shown, in the bottom row that of the unaddressed substrate (-P-). All cleavage experiments were done with 8 nM DNA and 8 nM enzyme. ( C ) Comparison of the cleavage rates of an unaddressed substrate by selected AvrBs3-PvuII fusion proteins (as indicated) under physiological ionic strength: 143 mM (20 mM Tris-Ac, 120 mM K-Ac, 1 mM Mg-Ac, pH 7.5). The experiments were done with an excess of enzyme, the TALE-scPvuII fusion protein (top, 60 nM enzyme, 6 nM DNA) shows a higher cleavage activity with an unaddressed substrate (-P-) than the homodimeric TALE-PvuII T46G fusion protein (bottom, 80 nM enzyme, 8 nM DNA). See the appearance of nicked and linearized DNA with AvrBs3-28-L-scPvuII T46G . There is no nicking or cleavage detectable of the unaddressed substrate with AvrBs3-28-L-PvuII T46G . oc, open circle; lin, linearized; sc, supercoiled.

    Techniques Used: Activity Assay

    Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins on AvrBs3 and AvrBs4 substrates. ( A ) Specificity of cleavage analyzed with the T3-6bp-P-6bp-T3 substrate and the T4-6bp-P-6bp-T4 substrate which differ in 11 (8, respectively, considering the degeneracy of the TALE recognition code) out of 19 positions from the AvrBs3 target site. No nicking or cleavage of the AvrBs4 substrate (8 nM) by AvrBs3-28-L-PvuII T46G (8 nM) could be detected. ( B ) Cleavage of a “half-site” substrate by AvrBs3-28-L-PvuII T46G . The “half-site” substrate is a bipartite substrate consisting of an AvrBs3 recognition site and a PvuII recognition site (T3-6bp-P). The sc plasmid (8 nM) with the “half-site” was incubated with an equimolar concentration of AvrBs3-28-L-PvuII T46G (8 nM). The assay was done under physiological ionic strength and in competition with a 32 nM PCR fragment (unP) with one unaddressed PvuII site (-P-). Whereas the “half-site” substrate is cleaved almost to completion, the unaddressed PCR fragment is not cleaved at all. ( C ) The effect of the distance of the AvrBs3 and the PvuII site on the rate of DNA cleavage by various AvrBs3-PvuII fusion proteins. 20 nM radioactively labelled PCR fragments with 2 (T3-2-P-2-T3), 4 (T3-4-P-4-T3), 6 (T3-6-P-6-T3) and 8 (T3-8-P-8-T3) bp between the AvrBs3 and the PvuII site were incubated with 20 nM AvrBs3-28-L-PvuII T46G , AvrBs3-28-PvuII T46G and AvrBs3-L-PvuII T46G for 60 min.
    Figure Legend Snippet: Analysis of the cleavage activity of AvrBs3-PvuII fusion proteins on AvrBs3 and AvrBs4 substrates. ( A ) Specificity of cleavage analyzed with the T3-6bp-P-6bp-T3 substrate and the T4-6bp-P-6bp-T4 substrate which differ in 11 (8, respectively, considering the degeneracy of the TALE recognition code) out of 19 positions from the AvrBs3 target site. No nicking or cleavage of the AvrBs4 substrate (8 nM) by AvrBs3-28-L-PvuII T46G (8 nM) could be detected. ( B ) Cleavage of a “half-site” substrate by AvrBs3-28-L-PvuII T46G . The “half-site” substrate is a bipartite substrate consisting of an AvrBs3 recognition site and a PvuII recognition site (T3-6bp-P). The sc plasmid (8 nM) with the “half-site” was incubated with an equimolar concentration of AvrBs3-28-L-PvuII T46G (8 nM). The assay was done under physiological ionic strength and in competition with a 32 nM PCR fragment (unP) with one unaddressed PvuII site (-P-). Whereas the “half-site” substrate is cleaved almost to completion, the unaddressed PCR fragment is not cleaved at all. ( C ) The effect of the distance of the AvrBs3 and the PvuII site on the rate of DNA cleavage by various AvrBs3-PvuII fusion proteins. 20 nM radioactively labelled PCR fragments with 2 (T3-2-P-2-T3), 4 (T3-4-P-4-T3), 6 (T3-6-P-6-T3) and 8 (T3-8-P-8-T3) bp between the AvrBs3 and the PvuII site were incubated with 20 nM AvrBs3-28-L-PvuII T46G , AvrBs3-28-PvuII T46G and AvrBs3-L-PvuII T46G for 60 min.

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Concentration Assay, Polymerase Chain Reaction

    TALE-PvuII fusion proteins. ( A ) Scheme of the architecture of TALE–PvuII fusion proteins. Left: wtPvuII, a homodimer in which the DNA-binding module of a TALE protein is fused via a linker of defined length. Right: scPvuII, a monomeric nuclease in which the DNA-binding module of a TALE protein is fused via a linker of defined length. ( B ) Model of a TALE–wtPvuII fusion protein. The fusion protein is a dimer of identical subunits, each composed of a PvuII subunit and a TALE protein. This model was constructed by aligning the structures of the individual proteins [pdb 1pvi [ 74 ] and pdb 3ugm [ 76 ]] on a DNA composed of the PvuII recognition site and two TALE target sites up- and downstream of the PvuII recognition site, separated by 6 bp. The C-termini of the PvuII subunits and the N-termini of the TALE protein are separated by about 3 nm. This distance must be covered by a peptide linker of suitable length. The image was generated with PyMol.
    Figure Legend Snippet: TALE-PvuII fusion proteins. ( A ) Scheme of the architecture of TALE–PvuII fusion proteins. Left: wtPvuII, a homodimer in which the DNA-binding module of a TALE protein is fused via a linker of defined length. Right: scPvuII, a monomeric nuclease in which the DNA-binding module of a TALE protein is fused via a linker of defined length. ( B ) Model of a TALE–wtPvuII fusion protein. The fusion protein is a dimer of identical subunits, each composed of a PvuII subunit and a TALE protein. This model was constructed by aligning the structures of the individual proteins [pdb 1pvi [ 74 ] and pdb 3ugm [ 76 ]] on a DNA composed of the PvuII recognition site and two TALE target sites up- and downstream of the PvuII recognition site, separated by 6 bp. The C-termini of the PvuII subunits and the N-termini of the TALE protein are separated by about 3 nm. This distance must be covered by a peptide linker of suitable length. The image was generated with PyMol.

    Techniques Used: Binding Assay, Construct, Generated

    8) Product Images from "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii"

    Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27004-1

    Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.
    Figure Legend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

    Techniques Used: Sequencing, Transfection

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    New England Biolabs pvuii hf
    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using <t>HpaI</t> and <t>PvuII</t> ensured that any residual partial digest products
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    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans

    doi: 10.1073/pnas.1220813110

    Figure Lengend Snippet: De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Article Snippet: Aliquots of 25 μg DNA were digested with 240 U HpaI plus 240 U PvuII-HF (New England BioLabs) in 500 μL NEB4 buffer at 37 °C for 2.5 h. Digested DNA was recovered by ethanol precipitation, dissolved in 5 mM Tris-HCl, pH 7.5, and 15 μg DNA was loaded into a 2.5-cm-wide slot in a 40-cm-long 0.9% (wt/vol) SeaKem HGT (Lonza) agarose gel in 0.5 × tris-borate EDTA buffer containing 0.5 μg/mL ethidium bromide.

    Techniques: Mutagenesis, Amplification

    Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

    Journal: Nature Communications

    Article Title: Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

    doi: 10.1038/s41467-021-27004-1

    Figure Lengend Snippet: Symmetrical editing up- and downstream of the DNA double-stranded break (DSB). a , b Fate of SNPs carried on sense ( a ) and antisense ( b ) ssODNs through single-strand DNA incorporation (ssDI) and synthesis-dependent strand annealing (SDSA). c Illustration of SNPs introduced into FKB12 using ssODNs (grey), annotated with the position (top) relative to the centre of the Cas12a-induced staggered DSB (red dotted line) and the SNP base being introduced (top, brackets). Asterisk (*) marks the base used for normalization during EditR. d – i Homology-directed repair (HDR, i.e., ssODN-mediated editing or SSTR) obtained using either five ssODNs carrying one SNP each ( d , g , n = 3), one ssODN carrying all five SNPs ( e , h , n = 3), or two ssODNs carrying either all up- or downstream SNPs ( f , i , n = 3) using sense ( d – f ) or antisense ( g – i ) ssODNs. Colour-coded p values relate to the significance of SNP detection from the chromatogram background noise by EditR (i.e., SNPs above α = 0.05 are indistinguishable from background noise, p values inversely correlate with editing levels and sequencing quality). HDR values and SNP detection p values are in Supplementary Data 2 and 12 , respectively. Of all analysis of variance (ANOVA) tests applied to each panel ( d – i ) only ( d ) was significant at ( F (4,10) = 4.844), p = 0.020; post-hoc Tukey test reveals one significant comparison between SNPs −16 and 0, p = 0.023. Full ANOVA and post-hoc results in Supplementary Data 16 and 17 , respectively. j Illustration of the restriction sites ( Bfa I, Pvu II) carried on a single ssODN, with the distance shown (top) relative to the Cas12a-induced staggered DSB (red dotted line). k Fate of restriction sites through ssDI and SDSA; only sense ssODN illustrated. l Restriction digestion of DNA from cells transfected with sense ( n = 3) or antisense ( n = 1) ssODNs. Values normalized to the site on the ssODN 5′ (sense: Bfa I, antisense: Pvu II). Sense ssODN one-sided one-sample Student’s t test t (2) = −1.742, p = 0.112, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}{:}{\mu }_{{PvuII}}=1$$\end{document} H 0 : μ P v u I I = 1 , Shapiro–Wilk for Pvu II is p = 0.712. Gel images, band quantification and non-normalized digestion efficiencies in Supplementary Fig. 6 . Bars are mean averages. Error bars are standard deviations. Repeats are biological (separately grown cultures). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${H}_{0}$$\end{document} H 0 : null hypothesis. PAM: protospacer-adjacent motif.

    Article Snippet: DNA (1500 ng) was digested using either Bfa I or PvuII-HF (20 U, New England Biolabs) in CutSmart buffer (1×) in 20 µL reactions by incubating at 37 °C for 1 h. Immediately thereafter, reactions were resolved on an agarose gel (1.5%) stained with SYBR Safe (1×), imaged (UVP BioDoc-It), and quantified semi-quantitatively using ImageJ (v1.51j8, raw ImageJ values in Supplementary Data ).

    Techniques: Sequencing, Transfection

    High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.

    Journal: Nucleic Acids Research

    Article Title: A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

    doi: 10.1093/nar/gkab770

    Figure Lengend Snippet: High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.

    Article Snippet: Using the psoralen-mediated cross-linking conditions indicated in the previous step, around 70–100 units of PvuII-HF enzyme (New England Biolabs, #R3151S) are sufficient to obtain digestion of 10–15 μg of genomic DNA (7–10 units/μg of genomic DNA) in three to five hours at 37°C.

    Techniques: In Vivo